JP6615148B2 - Induction of IL-12 using immunotherapy - Google Patents

Description

Detailed Description of the Invention

[Field]
The present invention relates to a therapeutic method using immune cells. More specifically, the present invention relates to immune cell therapy that promotes IL-12 production in patients.
[background]
The most accurate, powerful and safe disease prevention and treatment mechanism known combines elements of both innate and adaptive immunity to eliminate a wide variety of foreign pathogens without medical intervention, A natural “sterile” immune response. The immune system is built to “remember” removed foreign antigens in order to quickly initiate an immune response upon reinfection. The immune system can recognize foreign antigens such as those found in viruses and bacteria, even in the immune system of cancer patients, and is sufficient to completely destroy and remove the foreign antigens from the body, A response to a foreign antigen can be initiated. The strength and specificity of this sterile immune response can be confirmed by large transplanted organs such as the kidney, liver, or heart while the self-tissue is not affected by the compromised and lowered immune system Can be confirmed in that it can be completely destroyed. Such a destructive effect of immunity against foreign antigens would be beneficial if it could be converted to escaped tumors and / or other antigens because the patient's immune response has been reduced.

  Immunotherapy is dedicated to developing methods for utilizing, inducing, and controlling immune responses against various infectious and non-infectious diseases, including cancer. A therapeutic vaccine is a type of immunotherapy built to educate the immune system. In patients with cancer, the vaccine is designed so that the patient's immune system recognizes tumor cells as foreign. When the immune system recognizes a tumor as a foreign pathogen, it theoretically elicits an immune response that causes the immune cells to destroy large tumors and cause the metastatic tumor cells to appear wherever they are in the body. It can be found and destroyed. After successful immunotherapy, the immune system's ability to “remember” the removed foreign cells prevents the immune system from receiving additional treatment, just as the immune system prevents the affliction of opportunistic infections. Any recurrent cancer cells could be removed without application.

  An individual's immune system response to a disease or pathogenic organism can be a Th1 response or a Th2 response. In the Th1 response, CD4 + T cells differentiate into Th1 cells, whereas in the Th2 response, CD4 + T cells differentiate into Th2 cells. This more widely used classification method is called Th1 / Th2 balance. Th1 cells promote cell-mediated immunity, while Th2 cells induce humoral immunity. Cellular immunity (Th1) induces natural killer cells (NK), T cells, and macrophages to attack abnormal cells and microorganisms at the site of infection. Humoral immunity (Th2) results in the production of antibodies that are used to neutralize invading foreign bodies. In general, differentiation of CD4 + T cells into Th2 has been shown to be associated with cancer progression in many human and animal cancer studies, whereas Th1 differentiation is associated with tumor shrinkage and anti-tumor immunity. There is a correlation.

An individual's immune response, ie Th1 / Th2 balance, can be assessed by cytokine balance in the individual. Cytokines are small intracellular signaling protein molecules. The term cytokine is generally used as a collective term for various soluble protein groups and various peptide groups that act as regulators at concentrations from nanomolar to picomolar. Cytokines regulate the functional activity of individual cells and tissues, whether healthy or pathological. These proteins also directly regulate cell-cell interactions and regulate actions that take place in the extracellular environment. Interleukins are a group of cytokines involved in immune regulation and can be synthesized by various cells of the immune system. There are several interleukins such as IL-2, IL-4, IL-10, and IL-12, and each of these interleukins has a specific role in the immune system.

  Th1 cells produce type 1 cytokines that are involved in inflammatory responses. Type 1 cytokines include, for example, IL-2, IL-12, IL-15, IFN-gamma, TNF-alpha, TNF-beta, GM-CSF, and CC chemokines. Th2 cells produce type 2 cytokines that are involved in the humoral immune response. Type 2 cytokines include, for example, IL-4, IL-5, IL-6, IL-10, IL-13, and TGF-beta. The Th1 immune response and the Th2 immune response are counter-regulated to each other such that an increase in the type 1 response causes the type 2 response to be down-regulated and an increase in the type 2 response causes the type 1 response to be down-regulated. Fit.

  IL-12 is a heterodimer composed of p35 and p40 subunits. IL-12 is primarily produced by antigen presenting cells (APC). IL-12 can also be produced by monocytes and macrophages, dendritic cells, and B cells. IL-12 provides an immunomodulatory effect on T cells and natural killer cells. Endogenous IL-12 is known to be involved in producing an optimal Th1 response and can play an important role in cell-mediated immunity against intracellular pathogens.

  IL-12 has been the subject of intense research because it has been demonstrated to modulate important elements of the immune system and to have dramatic antitumor effects in laboratory studies and animal experiments. I came. IL-12 has been implicated, for example, in inhibiting the growth of human lung line carcinoma and acute myeloid leukemia. However, the use of exogenous IL-12 in treatment regimes has been limited due to its high toxicity to the human body.

1 is a graph illustrating IL-12 levels in a patient's plasma over a year. Allogeneic activated Th1 cells were administered to patients at various times using various means of administration.

Detailed Description of Exemplary Embodiments
The present invention relates to compositions and methods that lead to detectable levels of IL-12 in patient plasma. The present invention includes compositions that, when administered to a patient, lead to detectable levels of endogenous IL-12 in the patient's plasma without causing severe toxicity. Endogenous IL-12 can be surprisingly detected in cancer patients. The composition preferably comprises allogeneic activated T cells. Since T cells cannot produce IL-12, the T cell composition administered to the patient causes the production of IL-12 by the patient's own APC.

The invention also includes a method of inducing endogenous IL-12 production in a patient by the patient's own immune system. The method includes administering a composition of allogeneic material, preferably allogeneic activated T cells. The composition may be administered in a single dose or multiple doses. Preferably, allogeneic activated T cells are frequently administered at low doses. Allogeneic cells can be administered by intradermal, intravenous, or intralesional routes. The frequency is preferably greater than every 3 days. When these compositions are administered, even cancer patients can be induced by their own immune system to produce endogenous IL-12 in plasma at detectable levels. . Generally, IL-12 is not found in cancer patients because the tumor suppresses IL-12 expression. Surprisingly, the methods described herein are able to overcome this suppression and induce IL-12 expression in plasma over a long period of time, for example for months or even years. It is possible to create a sufficient environment. Furthermore, the presence of endogenous IL-12 in plasma does not cause the patient enough toxicity to administer exogenous IL-12 as a drug.

  Endogenous IL-12 means that IL-12 is synthesized within the patient by the patient's own immune system. Specifically, the IL-12 can be synthesized by the patient's antigen presenting cells (APC). APC can include monocytes and macrophages, dendritic cells, and B cells. Exogenous IL-12 means that IL-12 is not synthesized by the patient's own immune system. Exogenous IL-12 is IL-12 isolated from another individual and / or purified IL-12, or IL-12 expressed by a DNA construct containing the IL-12 gene (DNA construct), including.

  Advantageously, the endogenous production of endogenous IL-12 in a patient results in minimal or no toxicity to the patient. Patients may experience transient symptoms, such as transient flu-like symptoms. In general, when exogenous IL-12 has been administered to patients, its use in therapeutic settings has been limited by toxic effects. The method described herein is a method for promoting in vivo production of IL-12 such that the level of IL-12 in plasma can be detected systemically without causing toxicity, the method The ability of is amazing. As a result, by utilizing the patient's own immune system, it is possible to exploit the benefits arising from the presence of IL-12 for the purpose of shrinking and / or removing tumors and cancer cells.

  The use of these methods can be applied to the reduction and / or elimination of other diseases that respond favorably to the Th1 environment, specifically to IL-12. Such diseases include chronic viral and intracellular bacterial or mycotic, such as cancer, eg, hepatitis B, hepatitis C, HIV1, HIV2, HTLV1, HTLV2, HPV, Mycobacterium tuberculosis, periodontal disease, etc. Infectious diseases including bacterial diseases and allergic diseases such as atopic asthma are included. Furthermore, methods for promoting in vivo production of IL-12 can include an anti-aging effect by maintaining cellular immunity. The balance of Th1 cells to Th2 cells in healthy individuals decreases as part of the aging process, making older people more susceptible to infections and cancer. It is possible to increase the Th1 / Th2 ratio by promoting the production of endogenous IL-12, thereby protecting against vulnerability to disease.

  The composition of the invention generally comprises a foreign antigen, preferably an alloantigen. The composition comprises at least one Th1 cytokine and / or at least one DC effector molecule capable of inducing DC maturation to produce IL-12. A therapeutic composition generally comprises the at least one Th1 cytokine and / or the at least one DC effector molecule conjugated to a cognate antigen. The composition preferably comprises living allogeneic activated T cells capable of providing each component of the composition in a single cell type. In a preferred embodiment, it is activated to produce Th1 cytokines, such as interferon-gamma, tumor necrosis factor-alpha, and interleukin-2, and is a DC maturation effector molecule on the cell surface Live allogeneic Th1 cells expressing CD40L are used. Alternatively, the three components of the composition can be supplied from more than one cell type. For example, Th1 cytokines may be supplied from one cell type in one composition, alloantigens may be supplied from another cell type, and DC effector molecules may be supplied from a third cell type. Alternatively, one cell type may include any two of the above components, and the second cell type may include a third component. A cell type need not be alive as long as it provides a source of the necessary components of the composition.

  Alternatively, the components of the composition can be supplied from natural or bioengineered proteins. For example, recombinant or purified Th1 cytokines, DC mature molecules, or alloantigens may be used in combination or in combination with living cell components. The components of the composition can be combined on a “chip” or biodegradable platform. These components need not be administered to the patient simultaneously, but can be administered in any order.

  The alloantigen present in the therapeutic composition must be provided so that the antigen can be phagocytosed or can be donated to the immune system for the antigen to be processed and provided to T cells. The antigen may be a natural part of a living cell, or may be modified or bioengineered using molecular biotechnology techniques. The antigen can be soluble or immobilized to a surface, an intact part of a living organism or living cell, or a part of an attenuated organism. In a preferred embodiment, the allogeneic antigen is an allogeneic T cell, and in a more preferred embodiment is an allogeneic activated T cell.

  In one exemplary embodiment, the therapeutic composition comprises an alloantigen expressed on T cells. The T cell is preferably a CD4 + T cell, more preferably a Th1 cell. Th1 cells can be differentiated, expanded and activated in vitro from naive CD4 + precursor cells obtained from healthy blood donors. Preferably, the cells are in an activated state upon administration. Preferably, the cells are activated by cross-linking monoclonal antibodies to CD3 / CD28 surface molecules. Cross-linking is preferably caused by immobilizing CD3 / CD28 monoclonal antibody on the surface. Preferably, the surface is microbead particles or nanobead particles. These beads may be degradable beads. These cells can produce inflammatory Th1 cytokines in large amounts, and can express effector molecules such as CD40L on the cell surface. The effector molecule serves to promote the development of Th1 immunity by causing the production of endogenous IL-12.

  In a preferred embodiment, the therapeutic composition comprises activated allogeneic Th1 cells. These activated Th1 cells can be powerful inflammatory substances. These activated allogeneic Th1 cells and methods for their production are described, for example, in US Pat. Nos. 7,435,592, 7,678,572, 7,402,431, and 7,592, No. 431, which is incorporated herein by reference. Activated allogeneic Th1 cells are intentionally incompatible with patients.

  A variety of Th1 inflammatory cytokines can be included in the therapeutic composition. Examples of inflammatory Th1 cytokines include IL-1, IL-2, IL-6, IL-12, IL-15, IFN-gamma, TNF-alpha, TNF-beta, GM-CSF, and CC chemokines And TGF-beta, IL-4, or IL-10 is not included. The component of the cytokine may be a natural or recombinant cytokine, or it may be a bioengineered molecule constructed to interact with a cytokine receptor. Cytokines may be included directly in the therapeutic composition. Alternatively, the therapeutic composition may include living cells or other components that produce and secrete cytokines. Preferably, the cytokine is naturally provided through an activated cell source. The reason is that exogenous cytokines tend to be very toxic to patients while endogenous cytokines are not. In some exemplary embodiments, the therapeutic composition comprises T cells in an active state, and the T cells produce and secrete inflammatory Th1 cytokines, so that these in the therapeutic composition It can serve as a source of cytokines.

  The therapeutic composition may include one or more factors that cause maturation of immature DC. Specifically, maturation factors that promote DC1 cell maturation and IL-12 production result in interferon-gamma production and Th1 adaptive immunity. DCs can evolve from immature antigen capture cells to mature antigen-presenting T cells-priming cells, which are necessary to convert antigens to immunogens and initiate an immune response. It expresses cytokines, chemokines, and costimulatory molecules. The type of T cell-mediated immune response induced (Th1 vs. Th2) varies depending on the active signal received from the surrounding microenvironment. The ability of DCs to modulate immunity, such as anti-tumor immunity or anti-infection immunity, depends on the maturity of the DC to promote Th1 immunity. Human DCs are not a homogeneous population. In addition to inducing anti-tumor immunity, DC can also induce anergy or tolerance. DC are derived from CD34 + hematopoietic stem cells (HSC). Bone marrow dendritic cells (DC1) and plasmacytoid DCs (DC2) are two major subpopulations of human DCs whose characteristics vary greatly in phenotype, migration and function. DC1 cells are effective T cell stimulators and elicit tumor specific immune responses. CD11c + DC1 cells primarily induce Th1 differentiation, whereas DC2 cells that express the receptor for IL-3 (CD123) primarily promote Th2 responses. Both DC populations are significantly lower in cancer patients compared to healthy donors. DC1 cells produce IL-12 when mature, and DC2 cells produce IL-10.

  The production of cytokines such as IL-10 and IL-12 during the maturation process of DC affects the induction of DC into a Th1 or Th2 immune response. In addition to expressing antigen presenting molecules and costimulatory molecules at high levels, mature DCs must release large amounts of IL-12 in order to stimulate a Th1 immune response. Release of IL-10 prevents the DC maturation process by interfering with costimulatory molecule up-regulation and IL-12 production, which then limits the ability of DC to initiate a Th1 response .

  A variety of factors, including those described below, can induce maturation of DCs resulting in DC1, IL-12 producing cells following antigen uptake and antigen processing: antigens from all bacteria or bacteria (eg, lipopolysaccharide, LPS), inflammatory cytokines such as IFN-gamma, TNF-alpha, IL-1, GM-CSF, ligation of selective cell surface receptors (eg CD40), and viral products (eg double stranded RNA) . Fas binding in immature DC elicits, for example, both IL-1 beta and IFN-gamma maturation and release. CD40 ligation involves up-regulation of B7-1 / CD80 and B7-2 / CD86 costimulatory molecules, secretion of IL-12, and chemokines (eg, IL-8, MIP-1 alpha, MIP-1 beta). Promotes release.

  In some preferred embodiments, CD40L is included as a factor for DC maturation. It is within the scope of the present invention to include other factors that cause DC maturation. In some exemplary embodiments, the therapeutic composition comprises activated T cells that express a high density of CD40L on the surface. CD40L is a potent effector molecule for maturing DCs to produce IL-12.

  In one exemplary embodiment, the therapeutic composition comprises activated allogeneic T cells, at least one type I cytokine, and at least one factor that causes DC maturation. Compositions containing these components are described, for example, in pending US patent application Ser. No. 12 / 967,910, filed Dec. 14, 2010 and incorporated herein by reference. .

  In order to release tumor-associated antigens into the microenvironment, the therapeutic composition is administered intratumorally after excising part of the tumor cells, thereby providing a powerful adjuvant effect for DC to mature to the DC1 phenotype. Can be provided. The DC1 phenotype produces IL-12 and promotes the development of type 1 anti-tumor immunity and the down-regulation of tumor immune evasion mechanisms. Administration of the therapeutic composition can also be accomplished by other methods including, for example, intravenous administration, intradermal administration, intrathecal administration, intraperitoneal administration, intralesional administration, intrathoracic administration, and the like. Preferably, the composition is first administered transdermally because the skin is rich in immature DCs called Langerhans cells. For example, Langerhans cells ingest alloantigens in the presence of inflammatory Th1 cytokines such as interferon-gamma, tumor necrosis factor-alpha, IL-2, and GM-CSF, and DC maturation factors such as CD40L. Then, DC1 and IL-12 producing cells mature. These mature cells migrate to the lymph nodes and promote the development of Th1 immunity.

By intradermal injection of the composition, the patient can be “primed” to have immunity against the cognate antigen in the composition. Multiple intradermal injections can increase the number of Th1 memory cells specific for alloantigens in the patient's circulatory system, thereby changing the Th1 / Th2 balance. Injecting 1 × 10 ⁶ to 1 × 10 ⁷ allogeneic activated Th1 cells is the preferred intradermal dose, most preferably 1 × 10 ⁷ per ml of liquid It is. Intradermal administration is preferably repeated multiple times to increase the circulation of Th1 memory cells. The frequency of intradermal administration is preferably about 3-4 injections every 7 days, more preferably every 3-4 days.

In a preferred embodiment, intradermal administration is followed by intratumoral administration of the composition to create an in-situ vaccine. Intratumoral administration is preferably performed following excision of a number of tumor cells in the target lesion in-situ. Ablation is preferably performed using cryogenic (freeze ablation) or heat (radiotherapy), but can also be performed using a variety of methods including alcohol ablation, chemotherapy, and / or monoclonal antibody drugs. . A suitable intratumoral dose is between about 1 × 10 ⁷ and 1 × 10 ⁸ , most preferably about 3 × 10 ⁷ . The first intratumoral administration is injected immediately following ablation, and the second intratumoral administration is preferably injected within about 7 days, preferably within about 3-4 days after the first injection. . This ablation process followed by intratumoral injection of the composition can be repeated as necessary.

The aforementioned methods also preferably comprise compositions to cause activation of the host's (both congenital and adaptive) immune cells and their extravasation to inflammatory sites, including the location of the tumor. Including intravenous administration. Intravenous administration of the composition of allogeneic activated Th1 cells preferably includes about 1 × 10 ⁷ to 1 × 10 ⁹ , more preferably about 5 × 10 ⁷ to 1 × 10 ⁸ . This intravenous injection can be repeated several times, preferably once a month.

  The allogeneic Th1 cells of the composition preferably produce large amounts of type 1 cytokines, ie IL2, IFN-γ, TNF-alpha, and GM-CSF. The presence of inflammatory Th1 cytokines in the microenvironment in which immature DCs phagocytose and process antigens can help promote maturation into DC1, IL-12 producing cells. IL-12 can stimulate IFN-γ levels, which in turn can lead to the promotion of Th1 immunity. IFN-γ is an important type 1 cytokine necessary to promote type 1 anti-tumor immunity. IFN-γ can mediate anti-tumor effects by directly preventing tumor cell growth and inducing an anti-tumor response mediated by T cells. Secretion of IFN-γ independently contributes to the response of NK cells and can promote the response of NK cells activated by IL-12.

  Suitable agents comprising activated allogeneic Th1 cells can be obtained from purified precursors obtained from healthy sorted blood donors. The cells should be supplied in a sterile, low endotoxin dose, formulated for either intradermal or intratumoral injection, or intravenous infusion. The cells may also be formulated for intraperitoneal injection, intrapleural cavity injection, or epidural injection. The donor is preferably tested negative for HIV1, HIV2, HTLV1, HTLV2, HBV, HCV, RPR (syphilis) and the cells are negative for mycoplasma, EBV, and CMV It is preferable to be tested. In preferred embodiments, the activated allogeneic cells are HLA-incompatible with the patient.

  The methods of the invention generally relate to producing detectable levels of endogenous IL-12 in a patient's plasma. The method includes administering the composition of the invention in a manner that manipulates the patient's immune system to produce detectable levels of endogenous IL-12 in the patient's plasma. The methods described herein can increase the circulation of Th1 immune cells in cancer patients and shift the balance from the Th2 environment to the Th1 environment. In addition, the method may activate a component of the innate and adaptive immune response to elicit anti-tumor specific Th1 immunity and / or down-regulate tumor immune evasion to sustain Th1 cytokines A step of generating an environment may be included.

  The methods of the invention can include administering a composition comprising a foreign antigen to promote Th1 immunity of the patient against the foreign antigen. The method may also include excising all or part of a tumor that results in at least some tumor necrosis. Various methods can be used to necrotize a patient's tumor. The method may also include forming an inflammatory microenvironment proximate to a tumor necrosis site, ie, a tumor lesion site. Furthermore, the method can also include activating the patient's adaptive and innate immune cells to maintain a Th1 environment over time. In a preferred embodiment, an important element of the method includes the use of a drug or composition comprising activated allogeneic T cells, as described above.

  Since most human cancer patients generally exhibit polarized Th2 immunity, the purpose of this treatment method is generally to increase the number of circulating Th1 cells in cancer patients. . The number of circulating Th1 cells can be increased within the patient by administering to the cancer patient one of the therapeutic compositions containing foreign antigens described above.

  In an exemplary embodiment, the patient is administered activated allogeneic Th1 cells that are injected intradermally. In a preferred embodiment, intradermal injection is based on a weekly schedule of about 3 to 4 weeks once a week. In another preferred embodiment, the intradermal injection may be administered multiple times about every 3-4 days. Intradermal injections may be administered every other day or at intervals up to one year. Injection schedules should be formulated to improve the footprint of circulating Th1 memory cells. Alloantigens expressed in foreign cells can stimulate strong immune rejection. In addition, an inflammatory adjuvant environment is required to induce an immune response against alloantigens towards Th1 memory immunity by the presence of Th1 cytokines in the composition or by expression of Th1 cytokines by allogeneic cells. It is possible to provide. This makes it possible to increase the pool of Th1 memory cells circulating in the patient, specific for allogeneic antigens contained within allogeneic Th1 cells. Multiple doses can serve as a booster shot, thus increasing the number of circulating memory Th1 cells specific for alloantigens.

In some embodiments, following administration of allogeneic activated T cells, additional steps may be performed to improve patient response. These steps can include, for example, intratumoral administration of additional allogeneic activated T cells and excising a tumor that causes tumor necrosis. Allogeneic activated cells may additionally be administered intravenously. These methods are described in Har-Noy US Pat. No. 7,972,594, incorporated herein by reference.

  Administration of a therapeutic composition or agent using the methods described herein can promote systemic production of endogenous IL-12 in the patient by the patient's own immune system. The concentration of endogenous IL-12 in the patient is sufficient if IL-12 can be detected in the patient's plasma. The detectable level of IL-12 is endogenous and does not arise from IL-12 that may be present in the therapeutic composition. This is because, in general, in rejection induced by administration of allogeneic materials, the components of the composition are eliminated by the patient's immune system. In a preferred embodiment, the composition comprises T cells that are unable to produce IL-12. Thus, any IL-12 detected in the patient's plasma is a result of IL-12 produced by the patient's own immune system.

  Preferably, IL-12 is produced by the patient's immune cells, such as the patient's own monocytes, natural killer cells, and dendritic cells. These cells will mature under the influence of inflammatory or type I cytokines generated by administration of the compositions described herein.

  The concentration of IL-12 in the patient's plasma can vary, but is generally at least about 8000 pg / ml. The concentration of IL-12 in the patient's plasma is preferably between about 8000 pg / ml and 200,000 pg / ml. As described herein, the concentration of IL-12 detected in the patient's plasma does not cause toxicity problems. However, administration of exogenous IL-12 has been known to be harmful to patients. Patients who seroconvert to expression of IL-12 in plasma have a higher survival rate than patients who do not express IL-12 in their serum. Although the level of IL-12 may not correlate with survival, only the presence of IL-12 is crucial.

  In general, an increase in IL-12 is detected after a period of time has elapsed after administration of the composition. Preferably, IL-12 is detectable in plasma after administration of the therapeutic composition for about 3-4 weeks. After administration of the last composition, there can be a delay of about 90-120 days in IL-12 seroconversion.

  IL-12 in plasma can be detected using various methods. IL-12 has two subunits called p40 chain and p35 chain, and an antibody specific for p40 is suitable for detection. Several methods are available for detecting the presence of IL-12. For example, detection of IL-12 can include ELISA and cytokine bead arrays.

The methods described herein may be suitable for a variety of patients, including humans. The method may also be used for other mammals.
The invention also includes a method of treating a disease in a patient. The diseases can include not only diseases caused by pathogens but also cancerous tumors and hematological malignancies as described above. Other diseases that are susceptible to a Th1 response in a patient can also be treated using the methods described herein. The patient is administered the allogeneic composition according to the methods described herein. The patient's plasma is then monitored for the presence of IL-12. Detection of endogenous IL-12 can be indicative of the patient's immune response to the disease. Additional therapeutic compositions may be administered to maintain the level of IL-12, thereby maintaining the patient's immune response to the disease antigen.
[Example]
This study was conducted to monitor the level of IL-12 in the plasma of patients treated with allogeneic activated Th1 cells. Activated allogeneic Th1 cells and methods for their preparation are described in US Pat. No. 7,435,592. Activated allogeneic Th1 cells were intentionally incompatible with patients. Intradermal injection-Activated allogeneic Th1 cells were administered by intradermal injection. The cells were suspended in 1 ml at a density of 1 × 10 ⁷ cells / ml.

Intratumoral injection—Intratumoral injection was administered to the necrotic center of the resected tumor within 1 hour of resection.
Cryoablation was performed using the CryoCare-28 transcutaneous probe system (Endcare, CA, USA). This system used the Joule-Thomson effect to cool the freezer tip in a closed system. Depending on the gas coefficient and the size of the nozzle, different gas elements will cause different heat exchange phenomena in the area close to the nozzle. Argon gas was used for cooling (−187 ° C.) and helium was used for heating (67 ° C.).

  The planned target tumor lesion was identified and imaged under image guidance by CT. A sterile field was generated and local anesthesia was applied to the planned probe insertion site. The guide probe was inserted percutaneously and confirmed by CT to be within the target tumor lesion. One or two freeze-thaw cycles were performed. Depending on the size of the target tumor, one probe of 2 mm or 5 mm was used. The freezing time was about 5 to 20 minutes, depending on the formation of a “ice-ball” visible on CT. Thawing was achieved by injecting helium for a time equivalent to the freezing time before the second freezing process was started. This procedure requires excision of a sample of the tumor lesion, but does not require complete excision of the tumor, including the area where there is no tumor.

Following the second freezing cycle, the lesion could be cooled before injection of allogeneic activated Th1 cells.
Tables 1, 2, and 3 show the specific timing of treatment and the level of IL-12 in the patient's plasma over the days described. FIG. 1 is a graph illustrating the expression of IL-12 by the immune system of patients under study.

  Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention. .

Claims (11)

A composition for your Keru used in the manufacture of a medicament for inducing endogenous IL-12 in the patient's plasma,
The composition comprises
A first composition comprising about 1 × 10 ⁶ to about 1 × 10 ⁷ activated allogeneic Th1 cells; and
A second composition comprising about 1 × 10 ⁷ to about 1 × 10 ⁹ activated allogeneic Th1 cells,
Said first composition is administered intradermally at least twice;
The second composition is administered intravenously at least twice;

The composition is used to induce at least 8000 pg / ml to 200,000 pg / ml of endogenous IL-12 in a patient's plasma;
The composition is re-administered until plasma levels of the endogenous IL-12 are measured in the patient to induce an endogenous level of at least 8000 pg / ml to 200,000 pg / ml;
The composition wherein the activated allogeneic Th1 cells are activated by cross-linking of CD3 and CD28 . A composition for use according to claim 1, comprising:
For use, the composition further comprises one or more of IL-1, IL-2, IL-6, IL-8, IL-15, interferon-gamma, TNF-alpha, and GM-CSF. Composition. A composition for use according to claim 1, comprising:
The composition for use, wherein the activated Th1 cells express effector molecules CD40L and / or FasL on their surface. A composition for use according to any one of claims 1-3 .
A composition for use, wherein the components of the composition are fixed to a surface. A composition for use according to claim 4 , comprising:
A composition for use, wherein the surface is biodegradable. A composition for use according to any one of claims 1 to 5 , comprising
A composition for use, wherein the Th1 cells are packaged in a syringe or flexible container. A composition for use according to claim 1, comprising:
A composition for use, wherein the cells are suspended in a non-nutritive medium. A composition for use according to claim 1, comprising:
A composition for use wherein the level of endogenous IL-12 in the plasma of the patient is not toxic to the patient. A composition for use according to claim 1 , comprising:
A composition for use wherein the cross-linking is cross-linked by monoclonal antibodies such as anti-CD3 and anti-CD28 monoclonal antibodies. A composition for use according to any one of claims 1 to 9 , comprising
A composition for use, wherein the composition is administered more than every 3 days. A composition for use according to any one of claims 1 to 9 , comprising
A composition for use, wherein the composition is further administered by intratumoral administration.